Detection of mutations and variations occurring in DNA has become increasingly important in the fields of genetics, molecular diagnostics and cancer research. One type of variation, single-nucleotide polymorphism (SNP), has attracted much attention because it is the most common form of genetic variation. This type of single-base substitution in the genome occurs at a frequency of >1% in the human population. A recent estimate is that there is about one SNP per 1000 bp in human DNA. Other types of mutations involve insertion and deletion, and are found to occur at about one per 12 kb. The determination of SNPs can be used to study genetic linkages and for the diagnosis of diseases, especially cancer.
One way to fully characterize a mutation is to perform DNA sequencing on the sample. However, current DNA sequencing techniques are laborious and expensive. Large-scale DNA sequencing to detect mutations is also not efficient because a large portion of the sequences will give negative results considering that mutation is the exception. To save time and cost, rapid screening methods need to be developed to identify both known point mutations and unknown point mutations before any further characterization is undertaken.
The detection of mutation can be accomplished by using oligonucleotide arrays or DNA chips. Even though the number of analysis sites that can be packed into a small area array is very large, one must use multiple spots to span each mismatch (mutation). Using arrays, the match/mismatch discrimination is not entirely definitive, since different sequences have different melting temperatures. Ideally, one would have slightly different temperatures at each site within the array of sites. The other issue is time. In a representative mode of operation, the DNA is applied to the array and hybridization is carried out at 44° C. for 15 h at 40 rpm. The array of sites is then washed and stained before imaging. A third issue is that the DNA arrays are presently quite costly if one wants to span all possible mutations and probe scores of clinical samples at a time. Clearly, further development is needed to speed up the process and to make it more cost effective.
Mutations in DNA are readily detected by mass changes, such as by mass spectroscopic techniques. Substitutions are not so obvious because of the limited mass resolution of instruments that are reasonably accessible at present. Positional switches will not be detected at all because these do not result in a mass change.
A popular electrophoresis method to detect polymorphism is to rely on slight changes in conformations in single-stranded DNA (SSCP). This technique relies on subtle electrophoretic mobility differences between single strands of DNA that have different sequences. The mobility differences arise because, under the proper conditions, the different strands will have subtly different conformations in the separation medium. There are at least three important limitations to the sensitivity of SSCP analysis. First, the “mildly” denaturing condition is not well defined and may have to be optimized for each DNA region. This is because the conformation of each strand, and therefore any changes in conformation, is specific to a particular sequence. Therefore, the mobility differences will not be observed if the separation conditions are not optimized for each particular sequence in the sample. Second, visualization after the separation is complex. For example, the introduction of a radionucleotide probe or a fluorescence label into the DNA strand requires prior knowledge of the specific sequences of DNA regions around the point of mutation. Third, at present the assay is not reliable with fragments greater than around 200 bp and the sensitivity is only 60–95%.
For the analysis of double-stranded DNA, conformation-sensitive gel electrophoresis (CSGE) is possible. This approach is based on slight differences in conformations between the homoduplex and the heteroduplex DNA fragments. Just as discussed for SSCP above, the optimal gel and buffer conditions are particular to each sequence. Only when applied together with SSCP can the mutation detection rate approach 100%.
A different approach is to use denaturing gradient gel electrophoresis (DGGE). Separation is performed at a constant temperature but with a gel constructed to provide various degrees of denaturation along its length. If the sequence is known around the region probed, the mutation detection rate can reach 100%, but irreproducibility in creating identical gels makes implementation difficult. Also, it is often necessary to attach an artificial GC-rich sequence to the respective ends of the two strands to provide optimum separation.
Compared to SSCP or CSGE, DGGE can handle longer DNA fragments and is less time-consuming. An analog of DGGE is temperature-gradient gel electrophoresis (TGGE). In TGGE, instead of a denaturant gradient along the gel, a spatial or temporal temperature gradient is used to perform the same function. A simpler scheme is to apply constant denaturing capillary electrophoresis (CDCE). But this is again limited to defined mutations.
Capillary electrophoresis (CE) provides rapid analysis, a small sample requirement, and high sensitivity. It has been successfully used in many DNA analysis fields like sequencing and genotyping. Recently developed multiple-capillary arrays are ideal for high-throughput analysis. It is possible to detect mutations using CDCE with laser-induced fluorescence of covalent tags or with DGCE using a secondary polymer concentration gradient to refocus the sample band in addition to a denaturant gradient. The construction of gradients in the above techniques are tedious and hard to reproduce, especially for a capillary array.
The temperature of the separation medium within a capillary can be modified internally through ohmic heating by varying the electric potential across the capillary. Limitations of this technique include the narrow temperature range that can be achieved and the mutual dependence of the temperature and the electric field. This dependence is undesirable because the optimal separation conditions for a particular sample may not be achieved at an electric field consistent with heating the capillary to the required temperature.